Change in the electrophysiological parameters of human oocytes in the process of maturation outside the body]

The dynamics of electrophysiological parameters (membrane potential--MP and resistance--R) of the human oocytes was studied during their in vitro maturation. The relationship was established between the changes of electrophysiological parameters and meiotic phases. The average value of MP of diplotene oocytes was 22.0 +/- 0.3 mV and that of R 2.0 +/- 0.5 mO. The membrane depolarization was observed upon the meiotic reinitiation. The MP of diakinesis--metaphase I oocytes amounted to 10.0 +/- 0.3 mV and R to 10.0 +/- 0.5 mO. At metaphase II the temporary membrane repolarisation was noted in some cases which, in some oocytes, was replaced by the increasing hyperpolarisation on the 2--3rd day of cultivation. The input membrane resistance increased on the 2--3rd day of cultivation.     

Intestinal Na+/glucose cotransporter expressed in Xenopus oocytes is electrogenic.

The cloned rabbit intestinal Na+/glucose cotransporter was expressed in Xenopus oocytes, and transmembrane currents associated with this transporter were monitored using a two-electrode voltage clamp. Addition of D-glucose to a Na(+)-containing solution bathing these oocytes generated a current which was blocked by phlorizin. Water-injected control oocytes did not exhibit any currents under these conditions. The magnitude and shape of the currents were dependent on the extracellular glucose and Na+ concentrations and the membrane potential. At Vhold = -50 mV, the Km values for glucose and Na+ were 14 +/- 2 (N = 4) microM and 17 +/- 1 (N = 3) mM, respectively. These Km values and imax exhibited voltage dependence: increasing the membrane potential from -30 to -150 mV increased KGlcm and imax threefold and decreased KNam eightfold. The reversal potential (VR) of the phlorizin-sensitive, glucose-dependent current varied with log Nao+ (slope 46 +/- 6 [N = 9] mV). In the absence of sugar, a Na(+)-dependent, phlorizin-sensitive (Ki = 3 +/- 0.5 microM) current was detected only in RNA-injected oocytes. The amplitude of this current at -50 mV was 6 +/- 1% (N = 13) of the maximum current measured in the presence of D-glucose. The VR of this sugar-independent current varied with log Nao+ (slope 63 +/- 1 [N = 4] mV), indicating that the cotransporter may carry Na+ in the absence of sugar. We conclude that the Na+/glucose cotransporter is electrogenic and that investigations of currents associated with its operation can yield valuable insights into the mechanisms of solute translocation.     

Role of Ca2+-activated Cl- channels and MLCK in slow IJP in opossum esophageal smooth muscle

The possible contribution of Ca2+-activated Cl- channel [I(Cl(Ca))] and myosin light-chain kinase (MLCK) to nonadrenergic, noncholinergic slow inhibitory junction potentials (sIJP) was studied using conventional intracellular microelectrode recordings in circular smooth muscle of opossum esophageal body and guinea pig ileum perfused with Krebs solution containing atropine (3 microM), guanethidine (3 microM), and substance P (1 microM). In opossum esophageal circular smooth muscle, resting membrane potential (MP) was -51.9 +/- 0.7 mV (n = 89) with MP fluctuations of 1-3 mV. A single square-wave nerve stimulation of 0.5 ms duration and 80 V induced a sIJP with amplitude of 6.3 +/- 0.2 mV, half-amplitude duration of 635 +/- 19 ms, and rebound depolarization amplitude of 2.4 +/- 0.1 mV (n = 89). 9-Anthroic acid (A-9-C), niflumic acid (NFA), wortmannin, and 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9) abolished MP fluctuations, sIJP, and rebound depolarization in a concentration-dependent manner. A-9-C and NFA but not wortmannin and ML-9 hyperpolarized MP. In guinea pig ileal circular smooth muscle, nerve stimulation elicited an IJP composed of both fast (fIJP) and slow (sIJP) components, followed by rebound depolarization. NFA (200 microM) abolished sIJP and rebound depolarization but left the fIJP intact. These data suggest that in the tissues studied, activation of I(Cl(Ca)), which requires MLCK, contributes to resting MP, and that closing of I(Cl(Ca)) is responsible for sIJP.     

中华大蟾蜍卵母细胞质膜存在钙依赖性的氯离子通道

本文采用双微电极电压钳方法研究了中华大蟾蜍卵母细胞内源性电压门控型离子通道的成分及其生理特性。卵母细胞去极化至 -30 mV 及更正电压时,有一持续的电压依赖性外向电流出现。钾离子通道拮抗剂四乙基氯化氨(tetraethy-lammonium chloride, TEA, 10 mmol/L)和 4- 氨基吡啶(4-aminopyridine, 4-AP, 10 mmol/L)协同作用时,该电流只能被抑制到最大电流幅度的(23.4±0.72)%。但是,上述浓度的TEA和4-AP 与氯离子通道拮抗剂5- 硝基-2, 3- 苯酚丙胺苯甲酸盐 (5-nitro-2,3-phenypropylamino benzoate, NPPB, 30 μmol/L)、无钙 Ringer 氏液或钙离子通道拮抗剂维拉帕米(40 μmol/L)协同作用时,可分别将此外向电流抑制到最大电流幅度的(2.1±0.08)%、(2.2±0.04)% 和(3.1±0.15)%。结果表明,中华大蟾蜍卵母细胞质膜上除有钾离子电流之外,还存在钙依赖性的氯离子电流。     

Acetylcholine contracture of the fast muscle fibers in the lamprey, Lampetra fluviatilis]

Fast fibers of m. longitudinalis linguae respond to Ach by a transient contracture with a half-decay period of 3-5 sec. The threshold concentration of Ach is approximately 10(-7) g/ml. Ach contracture is based on even depolarization of the whole muscle membrane. Threshold level of the MP for the onset of contracture lies between --50 and --40 mV. In the presence of Ach depolarization decreases twofold within 40-70 sec. Relaxation is not due to the decrease of depolarization. Contraction--Ach concentration curve has a small slope; it reaches maximum at a concentration 10(-4) g/ml, which corresponds to the MP ca. -10mV. Equilibrium Ach potential is significantly shifted to depolarization as compared to that in frog muscles, being equal to +1+/-1.8 mV.     

Comparison of currents activated by noxious heat in rat and chicken primary sensory neurons

The vanilloid receptor 1 (VR1) gene is responsible for both capsaicin-, and low threshold (LT) noxious heat-sensitivity in mammalian primary sensory neurons. Although, birds lack capsaicin-sensitivity they express LT noxious heat-sensitivity. Here, we compared LT noxious heat-activated whole-cell currents produced by rat and chicken cultured dorsal root ganglion neurons in order to find out the similarities and differences in the LT noxious heat transduction mechanisms between the two species. No significant differences between rat and chicken neurons were found in the mean cell diameter of the LT noxious heat-sensitive cells (20.4+/-0.8 microm, n=19 and 20.6+/-0.6 microm, n=11, respectively) and the average threshold (45.7+/-0.5 degrees C, n=19 and 46.1+/-0.7 degrees C, n=11, respectively) and peak amplitude (-2.9+/-0.6 nA, n=19 and -2.1+/-0.6 nA, n=11, respectively) of the heat-evoked responses. The current-voltage curves of the responses both in rat and chicken cells reversed at the same range (-19.5+/-3.8 mV, n=4 and -15.5+/-1. 2 mV, n=3, respectively) and showed strong outward rectification at negative membrane potentials. While all LT noxious heat-sensitive rat cells responded to capsaicin, none of the chicken neurons produced detectable response to it. These findings suggest that a VR1 homologue which lacks to sequence for capsaicin-sensitivity is possibly the LT noxious heat transducer in chicken.     

Utilization of the growth phase of the first follicular wave for bovine oocyte collection improves blastocyst production

Characteristics of the follicle population and oocyte developmental competence at selected stages of follicular development were studied in cows with the aim to increase embryo production derived from oocytes collected by transvaginal aspiration. In Experiment 1, the growth phase before dominant follicle selection and the low dominant phase during dominant follicle regression were compared. Twenty-four cyclic Holstein cows, 4 to 6 yr of age, were divided into 2 groups. Animals were synchronized using two injections of prostaglandin F2alpha at 11 d intervals, and onset of estrus was determined (Day 0). Using ultrasonography, all follicles were counted and classified. Oocytes were aspirated once on Days I through 3 (Group 1, n=5) or Days 15 and 16 (Group 2, n=3) of the estrus cycle. The experiment was carried out in 3 replicates. In Experiment 2, the growth phase of the first follicular wave before dominant follicle selection was characterized in detail. Twelve cows of the same breed and age were divided into 3 groups. Their first estrus was synchronized as in Experiment 1, and each following estrus was induced using one injection of prostaglandin F2alpha administered 4 to 6 d after each aspiration performed. The ovaries were examined, and oocytes were collected repeatedly (total of 5 times per cow) on Days 1 (Group 3, n=4), 2 (Group 4, n=4) or 3 (Group 5, n=4) after estrus at 10 d intervals during a 40 d period. Viable oocytes were matured, fertilized and cultured using the standard methods. In Experiment 1, the mean numbers (+/-SD) of all follicles and of recovered and viable oocytes per donor were higher in Group 1 than in Group 2, but only the mean numbers (+/-SD) of larger follicles and recovered oocytes were statistically significant (8.0 +/- 0.6 and 6.2 +/- 0.6.vs. 3.3 +/- 0.5 and 2.8 +/- 0.2; P< 0.05). In Experiment 2, the percentage of larger follicles out of all visible follicles and the mean numbers (+/-SD) of larger follicles per donor were significantly higher (P<0.05) in Groups 4 (75.7 and 9.1 +/- 2.7) and 5 (66.3 and 8.5 +/- 2.9) when compared to Group 3 (27.9 and 3.8 +/- 0.8). The development rate of fertilized oocytes was significantly higher (P<0.05) in Groups 4 (27.8) and 5 (27.5) than in Group 3 (12.8). It can be concluded that it is possible to improve the efficiency of transvaginal aspiration and in vitro embryo production by utilization of the growth phase of the first follicular wave before dominant follicle selection.     

乙酰胆碱对蟾蜍背根神经节神经元膜电位的影响及离子机制

在离体灌流的蟾蜍背根神经节(DRG)标本上,用微电极进行胞内记录。在73个神经元中,依神经纤维的传导速度将神经元分为 A 型及 C 型,其中 A 型细胞67个,C 型6个,静息膜电位为-67.5±1.3mV((?)±SE)。当加4×10~(-4)—6×10~(-4)mol/L 乙酰胆碱(ACh),可观察到如下四种膜电位变化:1.超极化:幅值9.1±3.0mV((?)±SE,n=23);(2)去极化:幅值12.9±2.2mV((?)+SE,n=20);(3)双相反应(n=24):先超极化,后去极化,超极化幅值8.0±2.4mV((?)+SE),去极化幅值10.9±3.1mV((?)±SE);(4)无反应(n=6)。用阿托品(1.3×10~(-5)mol/L,n=23),或同时应用筒箭毒与六甲双铵(浓度均为1.4×10~(-5)mol/L,n=8)灌流,能分别阻断 ACh 引起的膜的超极化或去极化。ACh 引起超极化反应时膜电导平均增加13.8%,翻转电位值大约-96mV。四乙铵(TEA,20mmol/L)能使 ACh 的去极化幅值增加48.2±3.2%((?)±SE,n=6),超极化幅值减小79.4±4.3%((?)±SE,n=8)。MnCl_2(4mmol/L)使 ACh 的去极化及超极化幅值分别减小54.2±7.2%((?)±SE,n=5)及69.2±6.4%((?)±SE,n=14)。以上结果提示:ACh 引起的 DRG 神经细胞膜去极化反应由 N 型乙酰胆碱受体介导,而超极化反应由 Μ 型乙酰胆碱受体介导,前者可能包含了多种离子电导的改变,后者则可能与钾电导增加有关。     

离体培养小鼠神经细胞在有丝分裂过程中的膜电位变化（简报）

Primarily cultured dorsal root ganglion cells and olfactory bulb cells were dissected from 12 to 14-d-old fetal C57 BL/6 J mice. After the cells were cultured for about two weeks, the growing status of cells were observed and the membrane potentials(MP) were recorded. The results show that mean value of the MP in anaphase was -68 +/- 3.1 mV (SE, n = 3), same as in interphase: Beginning from telophase, the cell membrane in the equator contracted gradually to become a concave ditch, and the MP decreased obviously, the mean value was -23.3 +/- 3.3 mV (SE, n = 6). After this, MP recovered gradually, till it divided into two sister cells. MP which were recorded separately in two sister cells were similar. But usually MP did not recovered to their normal values immediately.     

Angiotensin II stimulates an endogenous response in Xenopus laevis ovarian follicles

While responses to angiotensin II have previously been induced in Xenopus laevis oocytes after injection of messenger RNA extracted from mammalian tissue, no endogenous responses of ovarian tissue to this hormone have been reported. Here we describe such an endogenous dose-dependent response to angiotensin II, detected by conventional electrophysiological techniques, in follicular oocytes. The ED50 of the response was estimated to be 0.15 +/- 0.07 microM (S.E.M.). Maximal depolarization, obtained at 1 microM angiotensin II, was 18.3 +/- 1.4 mV (n = 18, three experiments using oocytes from two toads, mean resting membrane potential = -42 +/- 2 mV). The response was absent from collagenase-treated oocytes or follicular oocytes treated with octanol, suggesting that the receptors are predominantly in the follicular layer surrounding the oocytes.     

Molecular determinants of inactivation within the I-II linker of alpha1E (CaV2.3) calcium channels

Voltage-dependent inactivation of CaV2.3 channels was investigated using point mutations in the beta-subunit-binding site (AID) of the I-II linker. The quintuple mutant alpha1E N381K + R384L + A385D + D388T + K389Q (NRADK-KLDTQ) inactivated like the wild-type alpha1E. In contrast, mutations of alpha1E at position R378 (position 5 of AID) into negatively charged residues Glu (E) or Asp (D) significantly slowed inactivation kinetics and shifted the voltage dependence of inactivation to more positive voltages. When co-injected with beta3, R378E inactivated with tau(inact) = 538 +/- 54 ms (n = 14) as compared with 74 +/- 4 ms (n = 21) for alpha1E (p < 0.001) with a mid-potential of inactivation E(0.5) = -44 +/- 2 mV (n = 10) for R378E as compared with E(0.5) = -64 +/- 3 mV (n = 9) for alpha1E. A series of mutations at position R378 suggest that positively charged residues could promote voltage-dependent inactivation. R378K behaved like the wild-type alpha1E whereas R378Q displayed intermediate inactivation kinetics. The reverse mutation E462R in the L-type alpha1C (CaV1.2) produced channels with inactivation properties comparable to alpha1E R378E. Hence, position 5 of the AID motif in the I-II linker could play a significant role in the inactivation of Ca(V)1.2 and CaV2.3 channels.     

The fertilization potential and associated membrane potential oscillations during the resumption of meiosis in the egg of the ascidian Phallusia mammillata.

The fertilization potential in Phallusia mammillata consisted of an initial rapid depolarization. This initial sperm-triggered depolarization was followed by a phase of membrane depolarization which was of either long or short duration, depending on the eggs. When of long duration, the phase of membrane depolarization was divided into two periods: the first one began with a plateau (Em = +20.2 +/- 1.1 mV; duration = 1.7 +/- 0.14 min) which was followed by a series of membrane potential oscillations (n = 3.1 +/- 0.25) lasting 2.4 +/- 0.2 min. The second period also began as a plateau (Em = approximately 0 mV; duration = 3.40 +/- 0.20 min) which was followed by a series of oscillations (n = 11.5 +/- 0.5) lasting 11.8 +/- 0.6 min, followed by a membrane repolarization. The second series of oscillations often continued rising from the resting potential value. In the eggs displaying a short duration of membrane depolarization, the second period of depolarization was shortened (lasting only 3.5 +/- 0.5 min) since it lacked the second plateau. In addition it displayed a smaller number of oscillations (n = 4.7 +/- 0.6). As a consequence of this shortening, the membrane repolarized sooner. After repolarization, the membrane displayed several potential oscillations that started from the repolarization level. Regardless of the length of the depolarized plateau phases, the total number of membrane oscillations and the time period during which they occurred were constant. Eggs displaying a long depolarization phase had 15.9 +/- 0.6 oscillations in a 19.5 +/- 0.6 min interval, while eggs having a short depolarization phase had 16.0 +/- 0.8 oscillations in a 18.1 +/- 0.3 min interval. The time period during which the potential oscillations occurred corresponded remarkably well with the time of the meiotic divisions: the formation of the first polar body was detected about 80 sec after the end of the first series of oscillations; the second polar body was extruded about 85 sec after the last membrane oscillation occurred.     

Assessment of viability and mitochondrial function of equine spermatozoa using double staining and flow cytometry

An objective double-staining method was developed to evaluate viability and mitochondrial function of stallion spermatozoa using flow cytometry. Sperm viability was assessed by propidium iodide (PI) exclusion, and mitochondrial function was measured by the intensity of rhodamine 123 (R123) fluorescence. Flow cytometry estimates of sperm viability measured by PI were equivalent (P > 0.05) to estimates made using Hoechst 33258 stain and fluorescent microscopy (% dead: 25 +/- 2.4 vs 21.5 +/- 3.5). The use of both PI and R123 was validated by addition of various proportions of freeze-shocked (membrane damaged) cells to viable spermatozoa. There was a high correlation (r(2) = 0.996) between increased PI positivestained (dead) cells and the number of membrane-damaged spermatozoa added (% dead: 29 +/- 0.4, 44 +/- 1.4, 58 +/- 0.9, 75 +/- 0.7 and 91+/- 0.25 vs 0, 25, 50, 75 and 100% damaged cells, respectively). Optimal mitochondrial activity (OMA), as assessed by R123 uptake, was also reduced proportionally (r(2) = 0.976) by the percentage of membrane-damaged cells added (% OMA: 48 +/- 0.6, 37 +/- 1.7, 29 +/- 0.5, 16 +/- 1, 3.8 +/- 1.3 vs 0, 25, 50, 75 and 100% damaged cells, respectively). The mitochondrial inhibitors rotenone and monensin significantly depressed optimal mitochondrial activity (P < 0.001), and there was a significant positive correlation (r(2) = 0.959) between the dose of inhibitors added and the population of sperm cells exhibiting minimal R123 staining (4 -/+ 0.9, 12 -/+ 1.6, 14 -/+ 0.1 and 28 -/+ 2% for treatments with 0, 0.5, 1 and 2 x 10(-5) M rotenone and 0, 0.5, 1, and 2 x 10(-4) M monensin, respectively). Finally, it was shown that treatments containing identical proportions of membrane-damaged cells yielded similar results in terms of viability and mitochondrial activity, irrespective of whether the staining procedure was single or double (P > 0.05). The results of the double-staining method revealed that the percentage of spermatozoa with optimally functioning mitochondria was significantly correlated with the percentage of viable (PI negative) sperm cells (r(2) = 0.998). Flow cytometric analyses using this staining procedure provides reliable and rapid (10,000 cells/min) qualitative assessment of stallion semen.     

The effects of oocyte storage and cumulus cell presence on canine zona penetration by domestic dog spermatozoa

Zona penetration assays (ZPAs) have been developed in numerous species to evaluate sperm fertilizing potential. Preservation methods to stockpile oocytes would be beneficial because of the difficulty in obtaining sufficient numbers of fresh oocytes. Using a canine ZPA, the objectives of this study were to evaluate: (1) two methods of storing canine oocytes (salt storage and intrafollicular cooling) and (2) the effects of cumulus cells on oocyte penetration. In experiment 1. oocytes from fresh ovaries were assigned at random to 3 categories: fresh control (FRE), salt storage in solution 1 (1.5 M MgCl2.6H2O; SS1) and salt storage in solution 2 (0.5 M (NH4)2SO4, 0.75 M MgCl2.6H2O, 0.2 mM ZnCl2; SS2). Each category was subdivided into two treatments: cumulus cells intact (intact) and cumulus cells removed (denuded), resulting in a total of six treatments with n > 15 oocytes per treatment for each ejaculate. Fresh (FRE) intact oocytes demonstrated greater sperm-oocyte interaction than other treatments, including FRE denuded oocytes (11.7 +/- 0.6 versus <4.1 +/- 0.5 sperm-oocyte and 94.9 versus <55.6% penetration; P < 0.01). Poor sperm-oocyte interaction was demonstrated with all salt-stored oocytes (< or = 1.6 +/- 0.2 sperm-oocyte and < 51% penetration), but was further attenuated in the absence of cumulus cells. In experiment 2, oocytes obtained from fresh (FRESH) or cooled (24 h COOL, 48 h COOL) ovaries were used with cumulus cells intact for a total of three treatments with n > 15 oocytes per treatment for each ejaculate. No significant difference was observed in sperm interaction between oocytes from fresh, 24 and 48 h COOL ovaries ( 12.3 +/- 0.5 to 13.1 +/- 0.4 sperm-oocyte and 92.2-97.7% penetration; P > 0.01). These results indicate that salt storage may cause damage to canine oocytes, subsequently impairing sperm penetration, whereas short-term intrafollicular cooling does not affect the oocyte's penetrability. Furthermore, greater sperm interaction in oocytes with an intact cumulus suggests a possible role for cumulus cells in canine gamete interaction.     

Internal Na+ and Mg2+ blockade of DRK1 (Kv2.1) potassium channels expressed in Xenopus oocytes. Inward rectification of a delayed rectifier

Delayed rectifier potassium channels were expressed in the membrane of Xenopus oocytes by injection of rat brain DRK1 (Kv2.1) cRNA, and currents were measured in cell-attached and inside-out patch configurations. In intact cells the current-voltage relationship displayed inward going rectification at potentials > +100 mV. Rectification was abolished by excision of membrane patches into solutions containing no Mg2+ or Na+ ions, but was restored by introducing Mg2+ or Na+ ions into the bath solution. At +50 mV, half- maximum blocking concentrations for Mg2+ and Na+ were 4.8 +/- 2.5 mM (n = 6) and 26 +/- 4 mM (n = 3) respectively. Increasing extracellular potassium concentration reduced the degree of rectification of intact cells. It is concluded that inward going rectification resulting from voltage-dependent block by internal cations can be observed with normally outwardly rectifying DRK1 channels.     

Redox studies of subunit interactivity in aerobic ribonucleotide reductase from Escherichia coli

Ribonucleotide reductase is a heterodimeric (alpha(2)beta(2)) allosteric enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides, an essential step in DNA biosynthesis and repair. In the enzymatically active form aerobic Escherichia coli ribonucleotide reductase is a complex of homodimeric R1 and R2 proteins. We use electrochemical studies of the dinuclear center to clarify the interplay of subunit interaction, the binding of allosteric effectors and substrate selectivity. Our studies show for the first time that electrochemical reduction of active R2 generates a distinct Met form of the diiron cluster, with a midpoint potential (-163 +/- 3 mV) different from that of R2(Met) produced by hydroxyurea (-115 +/- 2 mV). The redox potentials of both Met forms experience negative shifts when measured in the presence of R1, becoming -223 +/- 6 and -226 +/- 3 mV, respectively, demonstrating that R1-triggered conformational changes favor one configuration of the diiron cluster. We show that the association of a substrate analog and specificity effector (dGDP/dTTP or GMP/dTTP) with R1 regulates the redox properties of the diiron centers in R2. Their midpoint potential in the complex shifts to -192 +/- 2 mV for dGDP/dTTP and to -203 +/- 3 mV for GMP/dTTP. In contrast, reduction potential measurements show that the diiron cluster is not affected by ATP (0.35-1.45 mm) and dATP (0.3-0.6 mm) binding to R1. Binding of these effectors to the R1-R2 complex does not perturb the normal docking modes between R1 and R2 as similar redox shifts are observed for ATP or dATP associated with the R1-R2 complex.     

Collection of oocytes from cattle via follicular aspiration aided by ultrasound with or without gonadotropin pretreatment and in different reproductive stages

Ultrasound-guided follicular aspiration was performed on 29 Holstein-Friesian cows/heifers twice weekly at 3- to 4-d intervals over a period of 2 consecutive estrous cycles (total 42 d). For visualization of the ovaries and guidance of the aspiration needle, a 6.5 MHz fingertip probe on a 62 cm probe carrier was inserted into the vagina. The disposable aspiration needle was connected to a permanent rinse tubing system, thus ensuring minimum death of oocytes in the aspiration processs. After penetration of the vaginal wall, the needle was inserted into a follicle of the rectally fixed ovary. Cumulus oocyte complexes (COC) were aspirated at a pressure of 100 mm Hg. In the first experiment, the effect of an additional gonadotropin treatment 4 d prior to aspiration was investigated in 8 lactating cows. Following FSH-treatment, the number of aspirated follicles was higher (P < 0.05) than in the nontreated animals (10.6 +/- 0.7 vs 8.9 +/- 0.5). The number of recovered COC (7.0 +/- 0.6 vs 5.8 +/- 0.5), the recovery rate (COC per aspirated follicle) (66.6% vs 65.4%), the percentage of viable COC (56.8% vs 52.1%), the cleavage rate upon in vitro maturation and in vitro fertilization (56.7% vs 59.8%) as well as the rate of morula/blastocyst formation (3.8% vs 2.9%) were similar in both groups. In the second experiment, follicles were aspirated in 4 lactating cows, 6 dry cows, 4 pregnant cows (first 35 d of pregnancy), and 4 heifers. The average number of aspirated follicles and recovered COC was higher (P < 0.05) in the first 2 groups (10.6 +/- 0.6 and 9.3 +/- 0.7 follicles; 7.2 +/- 0.5 and 6.9 +/- 0.7 oocytes) than in trie 2 other treatment groups (7.3 +/- 0.5 and 8.1 +/- 0.5 follicles; 5.0 +/- 0.4 and 5.7 +/- 0.5 oocytes). The percentage of viable COC was higher (P < 0.05; 68.3%) in lactating animals than in all the other groups (49.7, 52.5 and 57.4%, respectively). Similarly, upon in vitro fertilization, cleavage rate was higher (P < 0.05; 63.4%) in lactating cows than in the other groups (43.7, 50.5, 55.1%, respectively). A total of 21.5, 22.7, 11.9 and 13.5%, respectively, in the 4 groups of the in vitro fertilized oocytes reached the morula and blastocyst stages. After transfer of a total of 48 embryos 22 pregnancies (45.8%) were established as detected on Day 65. We conclude that 1) repeated aspiration of viable COC at short intervals is possible, 2) additional FSH-treatment does not increase oocyte yields, and 3) viable blastocysts can be produced from cattle at various reproductive phases irrespective of the reproductive phase.     

Identification of a surface charged residue in the S3-S4 linker of the pacemaker (HCN) channel that influences activation gating

I(f), encoded by the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel family, is a key player in cardiac and neuronal pacing. Although HCN channels structurally resemble voltage-gated K(+) (Kv) channels, their structure-function correlation is much less clear. Here we probed the functional importance of the HCN1 S3-S4 linker by multiple substitutions of its residues. Neutralizing Glu(235), an acidic S3-S4 linker residue conserved in all hyperpolarization-activated channels, by Ala substitution produced a depolarizing activation shift (V(12) = -65.0 +/- 0.7 versus -70.6 +/- 0.7 mV for wild-type HCN1); the charge-reversed mutation E235R shifted activation even more positively (-56.2 +/- 0.5 mV). Increasing external Mg(2+) mimicked the progressive rightward shifts of E235A and E235R by gradually shifting activation (V(12) = 1 < 3 < 10 < 30 mm); Delta V(12) induced by 30 mm Mg(2+) was significantly attenuated for E235A (+7.9 +/- 1.2 versus +11.3 +/- 0.9 mV for wild-type HCN1) and E235R (+3.3 +/- 1.4 mV) channels, as if surface charges were already shielded. Consistent with an electrostatic role, the energetic changes associated with Delta V(12) resulting from various Glu(235) substitutions (i.e. Asp, Ala, Pro, His, Lys, and Arg) displayed a strong correlation with their charges (Delta Delta G = -2.1 +/- 0.3 kcal/mol/charge; r = 0.94). In contrast, D233E, D233A, D233G, and D233R did not alter activation gating. D233C (in C318S background) was also not externally accessible when probed with methanethiosulfonate ethylammonium (MTSEA). We conclude that the S3-S4 linker residue Glu(235) influences activation gating, probably by acting as a surface charge.     

Characteristics of the functional properties of the spinal cord motoneurons of old rats]

The mean membrane potential (MP) of old rats did not differ significantly from that in young mature rats ((58.4 +/- +/-1,4 mV and 56.6 +/- 1.26 mV, respectively). At the same time the frequency of detection of motor neurons with the MP OF 70 mV and more fell by 18.6%, and with the MP of 50-59 mV -increased by 14.2% in the old, in comparison with the young animals. The direct excitability threshold in old rats decreased (3.0 +/- 3-10(-9) in young mature and 2.0 +/- 0.2-10(-9) a in old rats; P less than 0.02). The number of discharges per 50 msec of the neuron poliarization reached 4-5, constituting 80-100 pulse/min. When determined by the first two intervals the action potential frequency reached 125 pulse/sec, and in the young mature rats--over 300 pulse/sec. The duration of antidromic spikes was increased (1.02 +/- 0.09 msec in young mature animals and 1.65 +/- 0.14 msec in the old animals; P less than 0.001). The antidromic spikes of the neurons in old mature rats, as a rule, had no delayed depolarization.     

中华大蟾蜍卵母细胞成熟过程中膜电位变化的实验分析

The full-grown oocytes obtained from toad (bufo bufo gargarizans) submitted in hibernation state or reared at 25-30 degrees C for several months, named hibernation oocyte or high temperature oocyte, had a membrane potential of -41.51 +/- 0.77 mV and -43.83 +/- 1.39 mV in Ringer's solution respectively. The hibernation oocytes underwent GVBD (germinal vesicle breakdown) and membrane depolarization at 19 +/- 1 degree C after progesterone stimulation. The membrane potential was about -20 mV at the period of GVBD, and -10 mV or so at 20 hours after the hormone treatment. However, the high temperature oocytes did not undergo GVBD, their membrane potential decreased before the fourth hour after treatment with progesterone and then recovered. If the hibernation oocytes were preincubated at 37-38 degrees C for 13 hours prior to the culture in the medium containing progesterone (10(-6)M, 37-38 degrees C), no GVBD was observed and the membrane depolarized before the fourth hour after treatment with progesterone then recovered, but MPF was detectable in the cytoplasm (unpublished). Both GVBD and membrane depolarization appeared in the hibernation oocytes and high temperature oocytes after injection of MPF. The time required for the hibernation oocytes injected MPF to attain the membrane potential about -20 mV was 4 hours earlier than that of progesterone treatment. It was just the time required for the appearance of MPF in the cytoplasm of oocytes treated with the hormone. It was noticed in our precedent article that a factor which appeared in the cytoplasm of high temperature oocytes differed from MPF. The factor was called Hibernation Oocyte Mature Promoting Factor (HOMPF).(ABSTRACT TRUNCATED AT 250 WORDS)